Endogenous non-stimulatory peptides are important in aiding T cell activation by limiting quantities of antigen. This project will determine the molecular basis for the role of non- stimulatory MHC-peptide complexes. The importance of CD8 or TCR interaction with a non-stimulatory or positive selecting MHCp, compared to its binding to the antigenic MHCp will be determined using single-chain MHC class I-peptide molecules, where the peptide is fixed, and the CD8 or TCR binding site can be mutated. Induction of interaction between TCR and CD8 during recognition by antigen plus non-stimulatory peptides will be measured using FRET microscopy. FRET will also be used to determine whether the non-stimulatory and antigenic MHCp are brought close together during antigen recognition. We will measure early signaling responses to the endogenous, non- stimulatory or positive selecting peptides in the presence or absence of small amounts of antigen, to understand how the T cells can be pre-stimulated by these ligands. The relative importance of CD8 binding to non-stimulatory MHCp itself (i.e. the adhesion function of CD8) versus its importance in concentrating the kinase Lck, will be measured. Whether phosphorylation of other TCRs than those that bind to the antigenic MHCp occurs will be tested using a FRET biosensor. Two photon microscopy of T cells will be used to understand how T cell behavior alters with the affinity of the ligand for the TCR and with the presence or absence of non-stimulatory pMHC. Transgenic mice that express fluorescent CD8 molecules will be used to investigate cell surface dynamics of CD8 during migration and antigen recognition in situ.